Microporous membranes have been demonstrated to have utility in a wide variety of applications. As such, numerous processes have been developed to produce such membranes. For example, U.S. Pat. No. 3,876,738 described a process for preparing a microporous membrane by quenching a solution of a film forming polymer in a non-solvent system for the polymer. European Patent Application No. 0 005 536 describes a similar process.
Commercially available microporous membranes, comprising for example nylon, are available from Pall Corporation, Glen Cove, N.Y. under the trademark ULTIPOR N.sub.66. Additionally, microporous membranes made of cellulose acetate, cellulose nitrate or mixtures thereof are widely available from a variety of sources. Other membranes, comprising yvinylidene fluoride, (PVDF), are available under the trademark Durapore.RTM. (Millipore Corporation, Bedford, Mass.). The nylon and nitrocellulose membranes exhibit hydrophilic properties, while the PVDF membranes are hydrophobic. It is possible, however, to coat the PVDF membranes with materials which render them hydrophilic. These hydrophilic Durapore.RTM. membranes are also available from the Millipore Corporation.
For certain applications, notably filtration and macromolecular transfer, it has been suggested that the performance of the material could be increased by providing an ionic functional group attached to the membrane surface which would serve to provide a fixed formal positive charge to the membrane. Such charge-modified membranes have been suggested for macromolecular transfer applications (e.g., DNA and blotting) in U.S. Pat. Nos. 4,512,896 and 4,601,828. Additionally, charge-modified membranes have been suggested for use as filtration materials in U.S. Pat. Nos. 4,473,474 and 4,673,504. In each of these, however, the invention is limited to methods for charge-modifying hydrophilic membranes and the use of the same. In fact, the latter two patents each provide an example describing unsuccessful attempts to charge-modify hydrophobic membranes. These attempts led to the conclusion in each of the patents that hydrophobic polymer membranes were not amenable to charge-modification by the methods attempted and described.
As such, the charge-modified microporous membranes used for macromolecular blotting and filtration applications have utilized hydrophilic membranes as starting materials.
The term "macromolecular blotting" as used herein refers to processes for transferring biological macromolecules such as nucleic acids and proteins from electrophoresis gels to some type of immobilizing matrix. Historically, nitrocellulose was used as a suitable blotting matrix. Of particular importance is nucleic acid blotting such as DNA blotting. A variety of DNA blotting techniques have been developed. The most common is referred to as "Southern Blotting". In this technique, DNA fragments are separated by chromatographic techniques and then denatured while still in the gel. The gel is neutralized and placed between wicking paper which is in contact with a buffer reservoir. Nitrocellulose is then placed on top of the gel and dry blotting papers are placed on top of the nitrocellulose. As the buffer flows into the gel, DNA is eluted and binds to the nitrocellulose, thereby transferring the DNA fragment pattern onto the nitrocellulose. The fragment pattern can then be detected using hybridization techniques employing labelled nucleic acids which are complementary to the specific bound fragments.
Since the development of the Southern blotting technique, a number of variations and improvements on the technique have been developed. For example, if the blotting paper is derivatized with diazobenzyloxymethyl groups, thereby forming a material commonly referred to as DBM-paper, RNA and proteins can be covalently attached to the material. Aminophenylthioether coated papers activated to the diazo form, (DPT-paper), can also be used to bind DNA, RNA and proteins. Other immobilization methods have used high salt or alkaline conditions in efforts to improve binding of DNA, RNA and proteins.
Other attempts to improve the binding process have concentrated on the blotting substrate by replacing nitrocellulose, for example, with other hydrophilic materials such as Nylon 66. Additionally, U.S. Pat. Nos. 4,512,896 and 4,673,504, previously described, suggest other materials such as hydrophilic PVDF for use as blotting substrates. These substrates, however, are again limited to producing hydrophilic materials which have been charge-modified. While these materials are an improvement over nitrocellulose, DBM-paper and DPT-paper, their charge retention, during hybridization and recycling, and performance under alkaline conditions could still be improved upon.